Protective epitopes of adenyl cyclase-haemolysin (AC-Hly), their application to the treatment or to the prevention of bordetella infections

ABSTRACT

Purified nucleic acids comprising a nucleotide sequence encoding an immunogenic polypeptide of adenyl cyclase-haemolysin (AC-Hly), which induces formation of protective antibodies against an infection by a bacteria selected from the group consisting of  B. pertussis, B. parapertussis,  and  B. bronchiseptica  when the nucleic acid or polypeptide is administered to a human or animal host. The nucleic acids are useful, for example, to induce a protective immune response in a host against infection by a bacteria selected from the group consisting of  B. pertussis, B. parapertussis,  and  B. bronchiseptica.

This application is a continuation of application Ser. No. 09/907,951,filed Jul. 19, 2001 (now U.S. Pat. No. 6,994,854), which is a divisionalof application Ser. No. 08/669,785, filed Jun. 27, 1996 (now U.S. Pat.No. 6,309,648). This application claims foreign priority to FR 95 07945,filed Jun. 30, 1995.

BACKGROUND OF THE INVENTION

The present application relates to amino acid sequences comprisingepitopes of adenyl cyclase—haemolysin from Bordetella. Adenylcyclase-haemolysin (AC-Hly) is one of the toxins participating in theBordetella infectious syndrome. AC-Hly is a bifunctional protein havingan adenyl cyclase activity and a haemolytic activity. It is secreted bythe bacterium. Its structural gene has been cloned and sequenced (GlaserP. et al., 1988, Molec. Microb. 2, 19-20). It is the case that thisprotein is part of the family of toxins termed “RTX” for “repeats intoxins” and exhibits homologies with haemolysin from Escherichia coliand Actinobacillus pleuropneumoniae, and the leucotoxins fromPasteurella haemolytica and Actinobacillus actinomycetemcomitans. Thisprotein, like PTX (pertussis toxin), is capable of penetrating intoeucaryotic cells such as the macrophages, of being activated bycalmodulin, of synthesizing large quantities of cAMP and of disruptingcellular functions (Coote J. 1992. FEMS Microbiol. Rev. 88:137-162).

The inventors have identified, within this sequence, various domainshaving the capacity to induce the formation of protective antibodiesagainst an infection by Bordetella, in particular by B. pertussis and/orB. parapertussis and/or B. bronchiseptica.

The subject of the invention is therefore amino acid sequences capableof entering into the composition of immunogenic compositions or ofprotective vaccines against Bordetella infections as well as antibodiesdirected against these amino acid sequences, capable of being used forexample in immunotherapy. Immunotherapy or serotherapy is especiallyapplicable in children, where appropriate in infants infected with B.pertussis, B. parapertussis or B. bronchiseptica. The invention proposesapplications in human medicine or in veterinary medicine.

The subject of the present invention is an amino acid sequence derivedfrom the polypeptide sequence of adenyl cyclase-haemolysin (AC-Hly),characterized in that it is capable of inducing the formation ofprotective antibodies against an infection by B. pertussis and/or B.parapertussis and/or B. bronchiseptica, and in that it is chosen fromthe following chains:

a) a sequence comprising the chain of amino acids situated approximatelybetween position 910, preferably 913, and the last C-terminal amino acidof the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO:2) orof a sequence corresponding to the preceding one in B. parapertussis orin B. bronchiseptica (SEQ ID NO: 4), the said sequence comprising amodification by addition of a fatty acid between amino acids 980approximately and 985 approximately, preferably at the level of aminoacid 983;

b) a sequence comprising a chain of 6 to 500 amino acids comprisingamino acids 385 to 400 of the polypeptide sequence of AC-Hly from B.pertussis (SEQ. ID NO: 2) or of a sequence corresponding to thepreceding one in B. parapertussis or in B. bronchiseptica (SEQ. ID NO:4);

c) a sequence comprising the chains of amino acids defined above in a)and b), the said chains being either contiguous, or combined via thechain of amino acids naturally present between the chains defined abovein a) and b) within the AC-Hly from B. pertussis, B. parapertussis or B.bronchiseptica, or combined via an antigenic sequence derived from aprotein different from AC-Hly, the amino acid sequence obtained having athree-dimensional conformation identical or analogous to that of thecorresponding polypeptide sequence of AC-Hly from B. pertussis, B.parapertussis or B. bronchiseptica.

The expression “amino acid sequence derived from the polypeptidesequence of AC-Hly” is understood to mean, within the framework of theinvention, a sequence whose amino acids are identical, by their natureor by their linkage, to those of the polypeptide sequence of AC-Hly orare, for some of them, substituted, deleted or added, in a manner suchthat the immunological properties of AC-Hly are conserved. Inparticular, such a sequence, derived from the polypeptide sequence ofAC-Hly is recognized by antibodies formed in a patient infected withBordetella, especially with B. pertussis, B. parapertussis or B.bronchiseptica.

It will be considered, within the framework of the present invention,that the structure or the conformation of AC-Hly is conserved, the aminoacid sequence of the invention then having a structure identical oranalogous to that of AC-Hly, when the said amino acid sequence iscapable, after immunization of a patient or an animal, of inducing aprotective immunity against Bordetella infections.

A sequence according to the invention can be obtained by proteolysis ofAC-Hly purified from Bordetella. Preferably, this sequence is obtainedby chemical synthesis or by genetic engineering techniques.

Thus, to produce an amino acid sequence according to the invention bygenetic engineering, plasmids carrying fragments of the CyaA gene willbe used.

By way of example, the chemical synthesis may be performed in automaticmachines of the Applied Biosystem type. It is also possible to use thetechnique of Betsou F. et al., 1993, Infect. Immun., 61:3583-3589.

In this manner, it will be possible to prepare a peptide correspondingto the chain of amino acids 385 to 400, or even 385 to 500 of AC-Hlyfrom B. pertussis or to the corresponding chain of AC-Hly from B.parapertussis or from B. bronchiseptica. The amino acid sequence (SEQ.ID NO: 2) as well as the nucleotide sequence (SEQ. ID NO: 1) of AC-Hlyfrom B. pertussis has been described in Glaser et al, 1988, Molec.Microb. 2-19-30 and is represented in FIG. 5. The amino acid sequence(SEQ. ID NO: 4) and the nucleotide sequence (SEQ. ID NO: 3) of AC-Hlyfrom B. bronchiseptica is represented in FIG. 6.

The amino acid sequence of AC-Hly from B. parapertussis and thenucleotide sequence encoding AC-Hly can be obtained by conventionaltechniques from the DNA from a strain of B. parapertussis, for examplethe strain No. 1 deposited at the CNCM on 2 December 1994 under the No.I-1498.

When an amino acid sequence according to the invention is produced bygenetic engineering from the DNA of the CyaA genes from B. pertussis, B.parapertussis or B. bronchiseptica, and when it comprises the amino acidin position 983 of AC-Hly from B. pertussis (SEQ. ID NO: 2) or acorresponding amino acid of AC-Hly from B. parapertussis or B.bronchiseptica (SEQ. ID NO: 4), care will be taken to ensure that thissequence is produced in a cellular host also expressing the CyaC genefrom the strains identified above. The expression of the CyaC geneallows the modification by addition of a fatty acid necessary toconserve in the amino acid sequence comprising residue 983. The additionof a fatty acid may, by way of example, be a palmitoylation.

Strains which can be used in order to have access to CyaA and CyaC genesare for example B. pertussis HAV deposited at the CNCM on 19 Oct. 1994under the No. I-1485, B. parapertussis I-1498 and B. bronchiseptica 973Sdeposited at the CNCM on 12 May 1989 under the No. I-858.

Moreover, the nucleotide sequences encoding AC-Hly from B. pertussis andfrom B. bronchiseptica are presented in FIGS. 5 and 6 (SEQ. ID NOS: 1and 3) respectively.

The nucleotide sequence of the CyaC gene which activates the CyaA genehas been described in the publication of Barry E. M. et al. (Journal ofBacteriology, January 1991, p. 720-726).

An advantageous sequence within the framework of the present inventionis the sequence corresponding to the regions termed modified region andrepeat region of AC-Hly (see FIG. 1).

When an antigenic sequence which is heterologous in relation to thepolypeptide sequence of AC-Hly is present, it may be a sequence of abacterial, viral or parasitic pathogenic organism in particular, againstwhich the formation of protective antibodies, for example, is sought.

The expression “protective antibodies against an infection by B.pertussis and/or B. bronchiseptica” is understood to mean antibodieswhich protect against the lethal and sublethal infections induced bythese bacteria, that is to say which protect against the disease and theinfection.

Advantageously, the amino acid sequences according to the invention,which are capable of inducing the formation of protective antibodiesagainst the infections designated above, are associated with factorsinvolved in the virulence of Bordetella, and are used for example in theform of polypeptide preparations or of bacterial extracts having thecapacity to induce protection against persistence of the bacteria in thehost.

An advantageous amino acid sequence within the framework of theinvention is a sequence characterized in that it is in the form of apolypeptide having a three-dimensional conformation identical oranalogous to that of the corresponding polypeptide sequence of AC-Hlyfrom B. pertussis or from B. parapertussis or from B. bronchiseptica, inthat it comprises the chain of amino acids situated between positions900 approximately, in particular 910, and the last C-terminal amino acidapproximately of the polypeptide sequence of AC-Hly from B. pertussis(SEQ. ID NO: 2) or of a sequence corresponding to the preceding one inB. parapertussis or in B. bronchiseptica (SEQ. ID NO: 4), the saidsequence comprising, in addition, a modification by addition of a fattyacid between amino acids 980 approximately and 985 approximately, inparticular at the level of amino acid 983.

This amino acid sequence comprises the modified region and the repeatregion of AC-Hly.

Another preferred amino acid sequence according to the invention is asequence characterized in that it is formed by the chain of amino acidsbetween the amino acid in position 385 approximately and approximatelythe last C-terminal amino acid of the polypeptide sequence of AC-Hlyfrom B. pertussis (SEQ. ID NO: 2) or of a sequence corresponding to thepreceding one in B. parapertussis or in B. bronchiseptica (SEQ. ID NO:4), the said sequence comprising a modification by addition of a fattyacid between amino acids 980 approximately and 985 approximately,preferably at the level of amino acid 983.

A subject of the invention is also an amino acid sequence enteringwithin the scope of the definitions given above, formed by the chain ofamino acids between the amino acid in position 385 approximately and theamino acid in position 400 approximately of the polypeptide sequence ofAC-Hly from B. pertussis (SEQ. ID NO: 2) or of a sequence correspondingto the preceding one in B. parapertussis or in B. bronchiseptica (SEQ.ID NO: 4).

This amino acid sequence advantageously comprises an epitope capable ofinducing the formation of protective antibodies against an infection byBordetella of the B. pertussis, B. parapertussis and B. bronchisepticatypes.

According to another embodiment of the invention, the amino acidsequence is characterized in that it is formed by the chain of aminoacids between the amino acid in position 385 approximately and the aminoacid in position 500 approximately of the polypeptide sequence of AC-Hlyfrom B. pertussis or of a sequence corresponding to the preceding one inB. parapertussis or in B. bronchiseptica.

Advantageously, this sequence comprising amino acids 385 to 400 or 385to 500 is presented in a conformation identical or analogous to theconformation which it possesses in the AC-Hly protein from Bordetella.

The subject of the invention is also amino acid sequences obtained bydeletion of polypeptide fragments from the sequence of AC-Hly fromBordetella, whether it is the sequence purified from the bacterium, andin particular from B. pertussis, B. parapertussis or B. bronchisepticaor whether it is a sequence obtained from a recombinant proteinr-AC-Hly.

An amino acid sequence thus obtained is advantageously the sequencecalled ΔCla corresponding to the AC-Hly chain modified by deletion of anΔCla fragment represented in FIG. 1, corresponding to the chain of aminoacids 827 to 887 of the polypeptide sequence of AC-Hly from B. pertussis(SEQ. ID NO: 2) or of a sequence corresponding to the preceding one inB. parapertussis (SEQ. ID NO: 4) or in B. bronchiseptica, the sequenceobtained comprising a modification by addition of a fatty acid betweenamino acids 980 approximately and 985 approximately, preferably at thelevel of amino acid 983.

Another fragment may be deleted from the AC-Hly sequence in order toform an amino acid sequence according to the invention; this is thepolypeptide fragment ΔH corresponding to the chain of amino acids 385 to827 of the polypeptide sequence of AC-Hly from B. pertussis (SEQ. ID.NO: 2) or of a sequence corresponding to the preceding one in B.parapertussis or in B. bronchiseptica (SEQ. ID NO: 4), the sequenceobtained comprising a modification by addition of a fatty acid betweenamino acids 980 approximately and 985 approximately, preferably at thelevel of amino acid 983.

The subject of the invention is also the amino acid sequence forming the“repeat region” of AC-Hly from Bordetella, between the amino acidresidues 1000 and 1600 approximately. The repeat region from B.bronchiseptica comprises one repeat less compared to the AC-Hly from B.pertussis. In this regard, the invention relates, for example, to thesequence comprising amino acids 1552 to 1592 of AC-Hly from B. pertussis(SEQ. ID NO: 2).

The invention also relates to the nucleotide sequences encoding theamino acid sequences described above.

The genes CyaA, CyaB and partially CyaD from B. bronchiseptica werecloned into the plasmid pFBD2 harboured by E. coli K12XLl and depositedat the CNCM on 21 Jun. 1995 under the No. I-1601.

The plasmid pFBD2 is obtained by insertion at the BamHI site of the 8 kbfragment from B. bronchiseptica carrying the genes CyaA, CyaB andpartially CyaD.

The subject of the invention is moreover polypeptide compositionscomprising sequences according to the invention originating from varioustypes of Bordetella. For example, an advantageous polypeptidecomposition comprises a sequence defined above from B. pertussis oranother sequence or several of these sequences from B. parapertussis.

Another polypeptide composition of the invention comprises, in addition,one or more sequences from B. bronchiseptica.

According to another embodiment of the invention, a polypeptidecomposition is characterized in that it comprises one or more sequencesdefined above from B. parapertussis and one or more sequences from B.bronchiseptica.

The subject of the invention is also immunogenic compositionscharacterized in that they comprise one or more sequences defined in thepreceding pages.

Advantageously for protection against infection by Bordetella and inparticular for protection against persistence of the bacteria in thehost, an immunogenic composition comprising the amino acid sequences ofthe invention may be characterized in that it comprises, in addition, abacterial extract containing the products of expression of the vrg genesfrom a strain of Bordetella chosen from B. pertussis, B. parapertussisor B. bronchiseptica or a portion of these expression products,sufficient to induce an immune response in a host to which the extractmight be administered.

In addition to the presence of various adhesins and toxins, Bordetellaeare characterized by regulation of the expression of the factorsinvolved in their virulence. In other words, Bordetellae undergo phasevariations and modulations.

Bordetellae, depending on their environment, can become “avirulent”,that is to say incapable of inducing lethality, an inflammatory reactionand pulmonary lesions in the murine model of respiratory infection. Theyundergo either a phase modulation or a phase variation. The phasevariation is observed at a frequency ranging from 10⁻³ to 10⁻⁶ and ispractically reversible. It results in a stoppage of the expression ofthe toxins and adhesins described above and in the expression of otherfactors still not well characterized (change of the Phase I “virulent”bacteria to Phase IV “avirulent” bacteria). The phase I and phase IVbacteria have been described by Lacey B. 1960, J. Hyg, 58:57-93. Thephase modulation, phenotypically similar to the phase variation, iscompletely reversible and results in a stoppage of the synthesis of theadhesins and toxins during environmental changes (composition of theculture medium, temperature and the like).

The phase variation and the phase modulation observed in Bordetella areunder the control of two regulatory genes, bvg A and bvg S (Arico B etal., 1989, Proc. Natl. Acad. Sci. USA, 86:6671-6675).

The bvg S gene encodes a protein sensitive to the external conditions.This protein modulates, by phosphorylation, the activity of the proteinencoded by the bvg A gene, which is, on the one hand, a positiveactivator of the transcription of the genes encoding the factors forvirulence (vag genes for “vir activated genes”) mentioned above (Uhl M.A. and Miller J, 1994, Proc. Natl. Acad. Sci. USA 91:1163-1167), and onthe other hand a repressor of the transcription of certain genes(Beattie D. T. et al., J. of Bacteriology, Jan. 93, p. 159-527). Thegenes whose expression is repressed are called vrg genes for “virrepressed genes” and are yet poorly characterized. It has however beenshown that the vrg 6 gene from B. pertussis encodes a protein having arole in the persistence of the bacterium in the host (Beatties D. etal., 1992, Infect. Imm. 60:571-577). In B. bronchiseptica, two proteinsencoded by the vrg genes have been characterized: they are proteins ofthe flagella type (B. bronchiseptica phase I is an immotile bacteriumwhich does not synthesize flagella but which synthesizes adhesins andtoxins, and B. bronchiseptica phase IV is a motile bacterium whichsynthesizes flagella).

The presence, in the immunogenic composition, of a bacterial extractcomprising the products of expression of the vrg genes would make itpossible to enhance the humoral and/or cellular immune response obtainedafter infection in vaccinated subjects and would also contribute to theprotection against the persistence of the bacterium.

The bacterial extract termed “vrg bacterial extract” which is inquestion above contains all the constituents of the external membrane ofa phase IV bacterium, that is to say of a bacterium not expressing thevag genes, including the LPS endotoxin. This endotoxin may however beeliminated or detoxified.

This extract may be present in the form of a suspension.

The “vrg” bacterial extracts used to carry out the invention arepreferably extracts termed “urea extracts”.

A “urea extract” is composed of a mixture of proteins expressed at thesurface of the bacterium and which are separated from the bacteriumafter incubation of the latter with 5 M urea. The “vrg” urea extractcontains several proteins as yet not characterized, the flagella and theLPS.

The use of urea extracts allows the production of a vaccine which ischeaper compared with a vaccine which would be obtained from theproteins contained in the extracts, in purified form.

In addition, the inventors have observed that the urea extracts used mayinduce a T type cell immuno response (lymphoproliferation), thusbehaving like the cellular vaccine used up until now.

On the contrary, exclusively acellular compositions are thought not toinduce a T response, a reaction which nevertheless occurs in the eventof infection.

The vrg urea extracts are respectively prepared from phase IV bacteria.Where appropriate, the phase IV bacteria are replaced with bacteriawhose bvg S gene is mutated such that the bacteria express only theproteins encoded by the vrg genes.

The preparation of these extracts is described in detail in theexperimental part.

Thus, the invention preferably relates to an immunogenic compositioncomprising both a vag urea extract from B. pertussis and a vrg ureaextract from B. pertussis.

An appropriate B. pertussis strain for the preparation of these extractsis the HAV strain entering within the framework of the invention anddeposited at the CNCM (Collection Nationale de Cultures deMicroorganismes in Paris), on 19 October 1994 under the No. I-1485. Toprepare the vag urea extract, the HAV strain can be used directly sinceit is a phase I strain.

On the other hand, the vrg urea extract is obtained from a phase IVstrain derived from the phase I strain for example by mutation of thebvg S gene from the bacterium or by culture of the said phase I strainin a medium containing only magnesium sulphate, so as to obtain theexpression of the B. pertussis vrg genes alone.

In the same manner and where appropriate to improve the immune responsein the host to which the immunogenic composition might be administered,the latter comprises, in addition, one or more adhesins or toxins fromB. pertussis, B. parapertussis or B. bronchiseptica, chosen from FHA,AGGs or PRN and PTX or a portion of these proteins, sufficient to inducean immune response in a host to which the extract might be administered.

Advantageously, the amino acid sequences derived from the AC-Hly toxinand, where appropriate, the proteins expressed by the vrg genes areobtained from the HAV strain deposited at the CNCM under the No. I-1485.

Likewise, the amino acid sequences from B. parapertussis used within theframework of the invention are obtained from strain No. 1 deposited atthe CNCM under the No. I-1498.

Moreover, the amino acid sequences from the bronchiseptica strain whichare used within the framework of the invention may be obtained from thestrain 9735 deposited at the CNCM under the No. I-858.

The references given above for the various Bordetella strains areindicated either to give access to the sequence of the proteins andwhere appropriate to reproduce this sequence by chemical synthesis, orto obtain the amino acid sequences of the invention by proteolysis ofthe proteins from Bordetella, or to give access to the DNA of thestrains and thus to allow the production of the amino acid sequences ofthe invention by genetic engineering techniques.

The subject of the invention is also a vaccine composition comprising,as active ingredient, an immunogenic composition defined above, incombination with a pharmaceutically acceptable vehicle and, whereappropriate, with an adjuvant.

The invention also relates to a process for the preparation ofmonoclonal antibodies recognizing the AC-Hly from B. pertussis, theAC-Hly from B. parapertussis and the AC-Hly from B. bronchiseptica,comprising the steps of:

immunizing an animal, for example a Balb/c mouse with a peptidecomprising the sequence of amino acids 385 to 400 of AC-Hly from B.pertussis (SEQ. ID NO: 2), the immunization being, where appropriate,carried out by means of repeated administrations of the peptide;

fusing the spleen cells of the immunized animal with myeloma cells toform a hybridoma;

culturing the hybridoma under conditions allowing the production ofantibodies;

recovering the antibodies directed against the sequence of amino acids385 to 400 of the AC-Hly from B. pertussis.

The process described above advantageously allows, by using for theimmunization an antigen specific for the AC-Hly from B. pertussis,monoclonal antibodies to be obtained which recognize both the AC-Hlyfrom B. pertussis and those from B. parapertussis and from B.bronchiseptica. In addition, such monoclonal antibodies advantageouslyhave protective capacities in relation to the infection of a human oranimal host by Bordetella of the B. pertussis, B. parapertussis and/orB. bronchiseptica type. Thus, the monoclonal antibodies obtained byusing the process described above can be used for the treatment ofpatients or animals infected with one or more strains of Bordetellachosen from B. pertussis, B. parapertussis or B. bronchiseptica.

In general, the subject of the invention is monoclonal antibodiescharacterized in that they recognize the sequence of amino acids 385 to400 of the AC-Hly from B. pertussis.

The invention relates, in this regard, to monoclonal antibodies whichrecognize an epitope comprising the sequence of amino acids 385-400 ofthe AC-Hly from B. pertussis, produced by the hybridoma B5-4 depositedat the CNCM on 19 Jun. 1996 under the No. I-1734.

Other advantageous monoclonal antibodies are produced against thesequence of the last 217 amino acids of the AC-Hly from B. pertussis andare produced by the hybridoma E17-21 deposited at the CNCM on 19 Jun.1996 under the No. I-1733.

The subject of the invention is also monoclonal antibodies directedagainst any of the amino acid sequences described above. Monoclonalantibodies directed specifically against the C-terminal sequence of theAC-Hly are among the advantageous antibodies of the invention. Specialantibodies are for example directed against a sequence derived from thecorresponding chain comprising the last 217 amino acids of the AC-Hlyfrom B. pertussis, especially the amino acids 1488 to 1705 of the AC-Hlyfrom B. pertussis or the amino acids 1489 to 1706 of the AC-Hly from B.bronchiseptica. These monoclonal antibodies may be prepared by a processanalogous to that which is described above for the monoclonal antibodiesobtained against the epitope contained in the sequence of amino acids385 to 400 approximately of the AC-Hly from B. pertussis.

The antibodies of the invention may be humanized, for example, byreplacing the hypervariable part of a human immunoglobulin, having noantibody function, with a hypervariable region of a monoclonalimmunoglobulin obtained by means of the technique described above.

Techniques which make it possible to humanize antibodies have forexample been described by Waldmann T., June 1991, Science, vol. 252, p.1657-1662; Winter G. et al., 1993, Immunology Today, vol. 14, No. 6, p.243-246; Carter et al., May 1992, Proc. Natl. Acad. Sci. USA, vol. 89,p. 4285-4289; Singer et al., 1 Apr. 1993, Journal of Immunology, vol.150, No. 7, p. 2844-2857.

The subject of the invention is also polyclonal sera as obtained byimmunization of an animal with an amino acid sequence corresponding tothe definitions given above and recovering the antibodies formed whichare capable of recognizing the sequences used for the immunization.

Where appropriate, the immunization comprises the administration, withthe amino acid sequences, of an adjuvant.

According to a specific embodiment of the invention, the immunization iscarried out with one or more amino acid sequences and with animmunogenic composition comprising various antigens of AC-Hly.

The invention also relates to a pharmaceutical composition comprising,as active ingredient, monoclonal antibodies according to the invention.

These antibodies, in particular the antibodies directed against thesequence comprising the amino acids 385 to 400 and/or the antibodiesdirected against the C-terminal part of the AC-Hly protein, can be usedas medicinal product in immuotherapy in a host infected with B.pertussis and/or B. parapertussis and/or B. bronchiseptica.

Use will be made for example of the monoclonal antibodies produced bythe hydridoma B5-4 deposited at the CNCM under the No. I-1734 or by thehybridoma E17-21 deposited at the CNCM under the No. I-1733.

The antibodies according to the invention may also be used as means foranalyzing the Bordetella strains collected. The monoclonal antibodiesdirected against the amino acid sequence 358 to 400 or against theC-terminal sequence of the AC-Hly protein are thus appropriate for theanalysis of the B. pertussis strains.

Also entering within the framework of the invention is a process for thein vitro detection of an infection by a Bordetella especially by B.pertussis, B. parapertussis or B. bronchiseptica, characterized in thata sample of biological liquid from a patient or from an animal capableof being infected by Bordetella is brought into contact with monoclonalantibodies defined above or with a polyclonal serum defined above and inthat an immunological reaction is detected between the said monoclonalor polyclonal antibodies and bacteria of the genus Bordetella when theyare present in the sample.

The detection of infection with a bacterium of the Bordetella genus,especially by B. pertussis, B. parapertussis and B. bronchiseptica, mayalso be carried out on a sample of a biological fluid from a patient orfrom an animal by bringing the sample tested into contact with an aminoacid sequence corresponding to the definitions above, followed by thedetection of an immunological reaction between the said amino acidsequences and the antibodies present in the sample tested.

The invention also relates to a pharmaceutical composition comprising,as active ingredient, nucleotide sequences described above.

The use of the nucleotide sequences in pharmaceutical compositions maybe implemented with reference to the technique described by Donnelly etal. (Nature Medecine, 1995, vol. 1. No. 6, p. 583-587).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the truncated proteins of the AC-Hlyused: the construction of the plasmids and the production of thecorresponding truncated proteins has been described in previouspublications from Sebo P. et al., 1991, Gene, 104:19-24 and Sebo P. etal., 1993, Mol. Microb. 9:999 1009. The numbers placed in the column“Mr” indicate the relative molecular masses of the toxins, calculatedfrom their deduced amino acid sequences. The numbers which follow thesymbol Δ in the name of the plasmid are the numbers for the first andthe last amino acid of the parts deleted from the reading frame of cyaA.The numbers which follow the symbol ΔC represent the missing C-terminalresidues. The cyaA alleles are coexpressed with the cyaC gene under thecontrol of signals for initiation of transcription and of translation ofthe lacZ gene identical to that present in the plasmid pCACT3 (Betsou F.et al., 1993, Infect. Immun. 61:3583-3589). ΔC1307 was produced fromPDIA 5240 (pACTΔC1307) in the presence of the cyaC gene expressed intrans from a compatible plasmid pPS4C (Betsou F. et al., 1993, Infect.Immun. 61:3583-3589 and Sebo P. et al., 1991, Gene, 104:19-24).

FIG. 2: Characterization of B. pertussis and of the r-AC-Hly protein:200 ng of purified B. pertussis and of r-AC-Hly were subjected toelectrophoresis on an 8-25% SDS-PAGE gel and the proteins were stainedwith Coomassie blue (A) or transferred to a HYBOND C-Super membrane andincubated with mixed anti-r-AC-Hly sera from 20 mice, 22 days after thevaccination (B) or with monoclonal antibodies specific for the AC-Hlyfrom B. pertussis (C) or a mixture of sera from infected children(infections confirmed by culture) (D) or a serum from mice infected withB. pertussis 18323 collected two weeks after the infection (E) or aserum from mice infected with B. pertussis collected two months afterthe infection (F). The immunodetection was performed withperoxidase-labelled rabbit anti-mouse antibodies. Line 1: B. pertussisAC-Hly; line 2: r-AC-Hly; line 3: ΔHR2; line 4: ΔH; line 5: ΔCla; line6: ΔC217; line 7: ΔC1307; the numbers indicate the molecular weightmarkers.

FIG. 3: Inhibition of the adenylate cyclase (A), haemolytic (B) andcytotoxic (C) activities of the AC-Hly from B. pertussis by antiseradirected against r-AC-Hly fragments obtained in various mice and by serafrom mice and children infected with B. pertussis: the AC-Hly wasincubated with various sera as described in the Materials and Methodspart. The bars indicate the standard deviation (n=4).

FIG. 4: Protective activities of r-AC-Hly and of its truncated proteins.3- to 4-week-old mice were immunized twice, at two week intervals, with15 μg of r-AC-Hly (Δ-Δ), ΔCla (▴-▴), ΔC1307 (●-●), or ΔHR2 ([ ]-[ ]),ΔC217 (▪-▪), ΔH (□-□) or B. pertussis AC-Hly (◯-◯) adsorbed ontoaluminium hydroxide, or with a buffer containing aluminium hydroxidealone as control (□-[ ]). They are infected via the intranasal route twoweeks later with 10⁵ CFU of B. pertussis 18323. The curves show thestandard geometric deviation (bars) for six mice per point.

FIG. 5: Nucelotide sequence of the gene encoding the AC-Hly from B.pertussis (SEQ. ID NO: 2) and the corresponding amino acid sequence.

FIG. 6: Nucelotide sequence of the gene encoding the AC-Hly from B.bronchiseptica (SEQ. ID NO: 3) and the corresponding amino acid sequence(SEQ. ID NO: 4).

EXAMPLES

Materials and Methods

Bacteria Strains, Plasmids and Growth Conditions:

The virulent strain B. pertussis 18323 (ATCC 9797) was cultured on aBordet Gengou agar medium supplemented with 15% defibrinated sheep blood(BG) at 36EC for 72 hours and again for 24 h. Subcultures in a liquidmedium were performed in a Stainer-Scholte medium (Stainer D. W. andScholte M. J., 1971, J. Gen. Microb., 63:211-220) for 20 h at 36EC untilan optical density at 650 nm of 1.0 is obtained (OD₆₂₀=1.0).

The plasmids used for the production of the recombinant protein r-AC-Hlyand its truncated derivatives in E. coli have been described in thefollowing publications: (Betsou F., P. Sebo and N. Guiso, 1993,CyaC-mediated activation is important not only for toxic but also forprotective activities of Bordetella pertussis adenylatecyclase-haemolysin, Infect. Immun. 61:3583-3589; Iwaki M., Ullmann A.and P. Sebo, 1995, “Oligomerization of the adenylate cyclase toxin ofBordetella pertussis: Evidence from in vitro complementation ofindividually inactive mutants”. Submitted; and Sebo P., P. Glaser, H.Sakamoto and A. Ullmann, 1991, High level synthesis of adenylate cyclasetoxin of Bordetella pertussis in a reconstructed Escherichia colisystem, Gene 104: 19-24.

The plasmids allow the production of the various proteins (FIG. 1) inthe presence of the activating protein CyaC. E. coli XL-1 Blue strains(Stratagene) comprising the respective plasmids were cultured at 37EC ona 2xYT medium containing 100 μg/l ampicillin at an optical density OD₆₀₀of 0.5 to 0.7, and induced in order to obtain the production of AC-Hlyby IPTG (1 mM) for four additional hours (Betsou F., P. Sebo, and N.Guiso, 1993, Infect. Immun. 61:3583-3589).

Tests of the Adenylate Cyclase, Haemolytic and Cytotoxic Activities

The adenylate cyclase activity was measured according to the proceduredescribed by Ladant D., C. Brezin, I. Crenon, J. M. Alonso, and N.Guiso, (1987, Bordetella pertussis adenylate cyclase: purification,characterization and radioimmunoassay, J. Biol. Chem. 261:16264-16269).One unit (U) of adenylate cyclase activity corresponds to 1 nmol of cAMPformed per minute at 30EC, at pH 8.0. The haemolytic and cytotoxicactivities of AC-Hly were determined at 37EC using washed sheeperythrocytes (109/ml) according to the description of Bellalou J., H.Sakamoto, D. Ladant, C. Geoffroy and A. Ullmann, (1990, Deletionsaffecting haemolytic and toxin activities of Bordetella pertussisadenylate cyclase, infect. Immun. 58: 3242-3247). The proteinconcentrations were determined by the method of Bradford (A rapid andsensitive method for the quantification of micrograms of protein,utilizing the principle of protein-dye binding. Anal. Biochem.72:248-257).

Tests of Inhibition of Adenylate Cyclase

The purified AC-Hly from B. pertussis was incubated at 100 U/ml in 50 mMTris-HCl at pH 7.6, with 0.2 mM CaCl₂ and 0.1 NP-40 with various seradiluted 100-fold for 18 to 20 h at 4EC; the post-incubation adenylatecyclase activity (IA) of the samples was measured. The AC (adenylatecyclase) activity after incubation with the serum from mice immunizedwith aluminium hydroxide alone (CA) was taken as reference for 100%activity (in other words 0% inhibition).

The percentage inhibition of the AC activity was calculated as follows:% inhibition=100%−(100%×IA/CA).Tests of Inhibition of the Haemolytic Activity

One unit (U) of toxin and 5 μl of the various sera were mixed in 1 ml of10 mM Tris-HCl at pH 8, with 2 mM CaCl₂, 150 mM NaCl and 1 μM bovinebrain calmodulin and preincubated for 20 min. at 4EC. Washed sheeperythrocytes (10⁹) were added and the remaining haemolytic activity wasdetermined after incubation for 3 h at room temperature. The unlysederythrocytes were recovered in the form of a pellet after centrifugationat 2000 rpm and the optical density of the haemoglobin released (RH)into the supernatants was measured at 541 nm. The haemolytic activity ofthe toxin incubated with the serum of mice immunized with only aluminiumhydroxide (CH) was taken as reference for 100% activity (0% inhibition).The sheep erythrocytes incubated without the toxin were used as controlfor nonspecific lysis. The percentage inhibition of the haemolyticactivity was calculated as follows: % inhibition=100%−(100%×RH/CH).

Tests of Inhibition of the Cytotoxic Activity

One unit (U) of toxin and 5 μl of various sera were preincubated at 4ECfor 20 minutes in a total volume of 1 ml of 10 mM Tris-HCl at pH 8, with2 mM CaCl₂, 150 mM NaCl, 5 mM glucose, 1 mg/ml BSA and 1 μM bovine braincalmodulin. Then 10⁹ washed sheep erythrocytes were added and theincubation was continued at 37EC for 30 minutes. In order to stop theactivity of the toxin, 50 μl of erythrocyte suspension were injectedinto 1 ml of 50 mM boiling sodium acetate at pH 5.2 and heated at 100ECfor 5 min. The quantity of cAMP formed in the lysed erythrocytes (IT)was determined by a standard ELISA method (Pradelles P. Grassi J.Chabardes D., Guiso N. 1989, Analytical Chemistry, 61:447-450). Thetoxin incubated at 4EC was used as negative control of cytotoxicity. Theactivity of the toxin incubated with the serum of mice immunized withaluminium hydroxide alone (CT) was considered as being the 100% activity(0% inhibition). The percentage inhibition of the cytotoxic activity wascalculated as being: % inhibition=100%−(100%×IT/CT).

Electrophoresis and Immunoblotting Methods

An SDS-PAGE electrophoresis was performed on an 8-25% ready-for-usepolyacrylamide gel for the PhastSystem (Pharmacia) and the separatedproteins were electrotransferred from the polyacrylamide gels to HybondC-Super membranes (Amersham). After blocking, the membranes wereincubated at a dilution of 10⁻³ with polyclonal sera at 4EC overnight.The immunochemical detection was performed using horseradishperoxidase-labelled sheep anti-mouse immunoglobulins and an enhancedchemiluminescence system (ECL-Amersham).

Production and Purification of AC-Hly.

The AC-Hly from B. pertussis, the r-AC-Hly (recombinant AC-Hly) producedfrom E. coli and the various recombinant truncated proteins wereextracted from bacteria with urea and purified on calmodulin affinitycolumns according to the method described by Guiso N., M. Szatanik andM. Rocandourt (1991, Protective activity of Bordetella adenylate cyclaseagainst bacterial colonization. Microb. Pathog. 11:423-431). Theenzymatic preparations were preserved in 8 M urea with 50 mM Tris-HCl atpH 8, with 0.2 mM CaCl₂, at −20EC. All the preparations were analysed inorder to determine their degree of purity by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

Active Immunization

The purified preparations of r-AC-Hly and of the truncated proteins wereadsorbed at 60 μg/ml on aluminium hydroxide (250 μg/ml). To carry out anactive immunization, 3- to 4-week-old female Balb/c mice (CERJ, StBerthevin, France) were treated by subcutaneous injection of 15 μg ofprotein antigens twice, at two weeks interval. The controls receivedonly buffer with aluminium hydroxide. The mice were then bled one weekfollowing the last injection, so as to allow access to the circulatingantibodies present. Sublethal respiratory infection was achieved twoweeks after the second immunization.

Intranasal Infection of the Mice

The B. pertussis strain was cultured on a Bordet Gengou BG medium for 48h according to the preceding description and the bacteria wereresuspended in 1% casamino acids. Sublethal quantities were administeredby intranasal injections of 50 μl of the bacterial suspensions. Theinfected mice were sacrificed by cervical dislocation 1 hour after theexposure (at a time designated by “day 0”) and on different days afterday 0 (6 mice during each stage). The lungs were collected asepticallyand homogenized in salt with a tissue grinder. Dilutions of thehomogeneous lung preparations were sampled on a BG medium and colonyforming units (cfu) were counted after 3 days of incubation at 36EC. Allthe experiments were performed at least twice and gave coherent results.

Preparation of the Immune Sera

To obtain the sera from the infected mice, 10 female Balb/c mice (4weeks old) were infected intranasally with 2×10⁵ virulent B. pertussisbacteria according to the technique described by Guiso N. Rocancourt, M.Szatanik and J. M. Alonso, (1989, Bordetella adenylate cyclase is avirulence associated factor and a protective antigen, Molec. Pathog.7:373-380). The mice were bled 14 days after the infection or ondifferent designated days following these 14 days.

The mouse polyclonal sera directed against the truncated derivatives ofAC-Hly were collected one week after the second injection given to themice with each of the antigens considered.

The serum of infected patients was prepared by mixing polyclonal immunesera from 10 selected nonvaccinated children infected with B. pertussis.These children were more than 8 months old, so as to exclude thepresence of maternal antibodies in them, and less than 2 years old so asto be certain of their clinical history.

Results

Immunological Properties of the r-AC-Hly Protein and of Its TruncatedVariants

It has been shown in the past that the modification of AC-Hly occurringthrough the CyaC protein from B. pertussis is essential for itsprotective activity (Betsou et al., CyaC-mediated activation isimportant not only for toxic but also for protective activities ofBordetella pertussis adenylate cyclase-haemolysin, Infect. Immun.,61:3583-3589). It was therefore important to determine if thismodification itself contributed to the formation of the protectiveepitope in its linear form or if this modification induced aconformational modification in the toxin, a modification required forthe presentation of the protective epitopes. Consequently, theimmunological and protective properties of a number of truncatedproteins were examined; the truncated proteins in question areschematically represented in FIG. 1.

These proteins were produced in E. coli in the presence of the CyaCprotein using the plasmids pCACT or pDIA so as to allow acylation byCyaC by means of fatty acid chains, of the constructs containing thesite of modification. As shown in FIG. 2A, the preparations purifiedfrom all the proteins contained a major polypeptide corresponding to theexpected molecular weight. These proteins were checked by a Western Blottest in order to verify their recognition by various sera. As shown inFIG. 2B, the serum produced against the r-AC-Hly protein recognized ther-AC-Hly protein and also recognized the AC-Hly protein purified from B.pertussis as well as all the truncated forms derived from r-AC-Hly. Inaccordance with FIG. 2A, the purified polypeptide preparationscomprising the total length of the protein, in other words thepreparations of AC-Hly, r-AC-Hly and the truncated polypeptides ΔCla andΔC217, also contained several fragments recognized by a polyclonal serumobtained against the purified protein r-AC-Hly (FIG. 2B). Monoclonalantibodies (FIG. 2) prepared specifically against the adenylate cyclasedomain of AC-Hly also recognized these fragments of AC-Hly, ΔCla andΔC217, showing that these fragments were proteolytic fragments truncatedin the C-terminal part and containing the AC domain which copurifiedwith AC-Hly on calmoduline-agarose. However, the monoclonal antibody didnot recognize the proteins ΔH or ΔHR2 which lacked residues 385 to 828and 1489 to 1706 respectively, but this antibody recognized the proteinΔC1307. Using these observations in particular, the inventorsestablished that the region of the molecule located between amino acids385 and 400 participated in the formation of an epitope.

Quite remarkably and contrary to what could happen with theanti-r-AC-Hly serum, the sera obtained from infected children and usedin the form of a mixture did not recognize the AC domain of AC-Hly(AC1307; line 7), and neither did they recognize the protein lacking the217 terminal residues of AC-Hly (ΔC217; line 6) nor the proteincontaining the last 217 residues but lacking hydrophobic regions, whichis modifed, and the major part of the repeat region of AC-Hly (ΔHR2,line 3). However, these human sera recognized the AC-Hly from B.pertussis and r-AC-Hly (FIG. 2D, lines 1 and 2) and the two truncatedproteins possessing modified and repeat regions (last 900 residues) ofAC-Hly (FIG. 2D, lines 4 and 5). An identical recognition pattern (FIG.2F) was obtained with mouse serum infected with B. pertussis 18323(reference strain) and collected rapidly after the infection (14 days).The sera collected long after the infection (35 days) recognized theC-terminal part of AC-Hly present in the protein ΔHR2 (FIG. 2F, line 3),whereas they continued not to recognize the AC domain (FIG. 2F, line 7)and the protein ΔC217 lacking only the last 217 residues (FIG. 2F, line6). These results strongly suggest that the anti-AC-Hly antibodiessynthesized after infection with B. pertussis are predominantly directedagainst the C-terminal region comprising the modification region and therepeat region of AC-Hly (last 900 residues). Furthermore, neither theprotein ΔC217 lacking the last 217 residues of AC-Hly, nor the proteinAHR2 which contained the last 217 residues were recognized by the immuneand murine sera, suggesting that these polyclonal sera recognize aspecific structure of the repeat region of AC-Hly, which structure isabolished by any of the two nonoverlapping deletions.

It is important to note that the sera from infected patients or micerecognized only the polypeptides stretching over the entire length ofthe protein (AC-Hly, r-AC-Hly, ΔCla and ΔH) but not their proteolyticfragments. The absence of recognition, by these sera, of the proteolysisproducts, which contain the AC domain and are cleaved in theirC-terminal part (see above FIG. 2C) indicates, in addition, that theregion of modification and the repeat region of AC-Hly (last 900residues) must be intact to be recognized by these sera.

Inhibition of the Adenylate Cyclase, Haemolytic and Cytotoxic Activitiesby Sera Directed Against Truncated Derivatives of AC-Hly

It was examined if sera from mice infected with B. pertussis or serafrom human patients infected with B. pertussis or alternativelypolyclonal sera directed against the various truncated forms ofr-AC-Hly, obtained by immunization with the purified truncated proteins,could specifically inhibit one of the activities of the toxin. Since acontrol was necessary prior to these experiments, the ELISA titres ofthe various sera were determined using the complete r-AC-Hly as coatingantigen for these tests. These titres were similar, with the exceptionof the titre obtained with the anti-ΔC1307 serum which did not recognizeAC-Hly in ELISA although it recognized it in a Western Blot test likethe other sera. As shown in FIG. 3A, the adenylate cyclase activity wasinhibited by all of the sera obtained after immunization of mice withpurified r-AC-Hly and with truncated derived proteins. However, none ofthe sera from the infected mice or from the infected human patientsinhibited the AC activity of AC-Hly under identical conditions. This isin agreement with the result obtained by the Western Blot analysis (seeabove) showing that these sera contain antibodies predominantly directedagainst the C-terminal part of AC-Hly (last 900 residues) and notagainst the AC domain. Indeed, the haemolytic activity of the proteinAC-Hly was inhibited by the sera from infected children and was lessstrongly inhibited by the sera from infected mice. However, a stronginhibition of the haemolytic activity was observed with the antiseraobtained by immunization with the proteins AC-Hly, r-AC-Hly, ΔH, ΔClaand ΔHR2 from B. pertussis (FIG. 3B). This inhibition was probably dueto the presence of specific antibodies against the C-terminal part ofAC-Hly. Indeed, the anti-ΔC1307 serum directed against the AC domain ofAC-Hly which lacked such antibodies did not inhibit the haemolyticactivity of AC-Hly. In a completely advantageous manner, the serumdirected against the protein ΔC217 lacking only the last 217 residuesdid not inhibit the haemolytic activity of AC-Hly at a measurable leveleither. This indicates that the last 217 residues are either the targetof the neutralizing antibodies or are involved in the formation of astructure which favours the synthesis of neutralizing antibodiesdirected against other parts of AC-Hly. Together, these resultsindicate, in addition, that the antibodies synthesized after infectionare mainly directed against the C-terminal haemolysin part of AC-Hly andare capable of neutralizing the haemolytic activity of this protein butnot the enzymatic activity of its N-terminal adenylate cyclase domain.As shown in FIG. 3C, a significant inhibition of the cytotoxic activityof AC-Hly was observed with sera obtained against intact AC-Hly andagainst the truncated proteins ΔH, ΔCla and ΔHR2 which all contain boththe AC domain and the last 217 residues. The antisera also neutralizedthe AC and haemolytic activities. On the contrary, the 2 sera whichinhibited either the haemolytic activity alone (for example the serafrom infected mice or patients) or the AC activity alone (theanti-ΔC1306 and anti-ΔC217 sera) did not inhibit the cytotoxic activityof AC-Hly. Thus, it appeared that the presence of antibodies against theAC domain and against the last 217 residues of AC-Hly may be required inorder to neutralize its cytotoxic activity.

Protective Activity of the Various Recombinant AC-Hly Constructs

In order to locate the epitopes of AC-Hly required to obtain aprotective activity, the protective activity of various purifiedtruncated proteins using the murine respiratory model was tested. Groupsof mice were immunized twice with aluminium hydroxide alone (control),or with purified truncated proteins adsorbed on aluminium hydroxide andthen these mice were brought into contact, via the intranasal route,with sublethal doses of virulent B. pertussis 18323 strains. This modelreflects the capacity of the bacteria to adhere, colonize, survive andmultiply in the respiratory system of mice. As shown in FIG. 4, thebacteria multiply rapidly in the lungs of the control mice for 6 daysafter infection, and then begin to be eliminated from the lungs. Nomultiplication of the bacteria was observed in the lungs of miceimmunized with the proteins AC-Hly or r-AC-Hly from B. pertussis andafter 3 days the number of bacteria began to increase (FIG. 4). Inagreement with previous observations (Betsou F., Sebo and N. Guiso,1993, CyaC-mediated activation is important not only for toxic but alsofor protective activities of Bordetella pertussis adenylatecyclase-haemolysin, Infect. Immun. 61:3583-3589), the protectiveefficacy of B. pertussis AC-Hly was higher than that of r-AC-Hly. Aprotection similar to that induced by r-AC-Hly was also obtained withthe protein ΔCla, suggesting that the part deleted in the protein ΔClabetween residues 827 and 887 was not essential for the induction ofprotective immunity. The protein ΔH, missing from residues 385 to 828,exhibited a weaker protective activity than ΔCla. No protection wasinduced by the protein ΔC1307 lacking the entire haemolysin part ofAC-Hly. In a more advantageous manner, the protein ΔC217 lacking thelast 217 residues and the protein ΔHR2 containing the last 217 residuesof AC-Hly did not make it possible to induce protection. The protectiveactivity is therefore correlated with the pattern of recognition of theindividual constructs by the sera from infected patients or frominfected mice.

Discussion

The antibodies directed against AC-Hly are usually present in the serafrom infected children (Arciniaga J. L., E. L. Hewlett, F. D. Johnson,A. Deforest, S. G. F. Wassilak, I. M. Onorato, C. R. Manclark and D. L.Burns, 1991, J. Infect. Dis. 163:135-142; and Guiso N., E. Grimprel, I.Anjak and P. Begue, 1993, Eur. J. Clin. Microbiol. and Infect. Dis. Inpress) and it has in the past been demonstrated that immunization withAC-Hly protects mice against bacterial colonization by Bordetella(Khelef, N., H. Sakamoto and N. Guiso, 1992, Microb. Pathog.12:227-235). The production of a set of truncated forms of recombinantAC-Hly has made it possible to carry out a study of the importance ofvarious domains of the protein in the induction of protective immunityby vaccination with AC-Hly. The results presented above show that theanti-AC-Hly antibodies, synthesized after infection by B. pertussis, arepredominantly directed against the modified domain and the repeat domainof AC-Hly (about 800 residues). Indeed, only the truncated forms ofAC-Hly possessing these in tact regions were recognized by sera frominfected mice and patients in a Western-Blot test and these proteinswere the only ones to exhibit the capacity of inducing protection inmice. This indicates that at least in the case of mice, a relativelylimited set of identical or overlapping epitopes may be advantageousboth for inducing the synthesis of anti-AC-Hly antibodies by theinfected subjects and for inducing the synthesis of protectiveantibodies obtained after vaccination with AC-Hly. The experimentsreported previously have shown that the elimination of the last 217residues of AC-Hly which are in the distal position relative to thesites of modification of the protein by the product of expression of theCyaC gene, at the level of the lysine residue 983, abolished therecognition of this protein (ΔC217) by sera from infected mice andpatients and also abolished its protective activity. However, this wasnot due to a defect in modification of the protein ΔC217 since thisprotein was acylated at the level of the fatty acid chains when it wasproduced in the presence of the protein CyaC. Furthermore, the 217residues per se were not recognized by the sera and did not show anyprotective activity when they were presented in the protein ΔHR2 which,for its part, lacked the modification site (lysine 983) and a largeproportion of the repeat region.

This led to the conclusion that both the last 217 residues and themodification region were important for the protective activity ofAC-Hly. Thus, interaction between the modified region and the last 217residues of AC-Hly would be required for the formation or the activityof the protective epitopes of AC-Hly. Surprisingly, neither the proteinAC217 missing from the last 217 residues of AC-Hly, nor the protein ΔHR2lacking from the modified region and from a large portion of the repeatsbut containing the last 217 residues were recognized by the polyclonalsera from infected patients and/or from mice (fresh serum). The antiseracould have a narrow specificity against a single epitope, or against asmall group of epitopes located at the breakpoint of the repeat regionspresent in the proteins ΔC217 and ΔHR2 (proline 1489). However, thispossibility does not appear highly probable with mixed polyclonalantisera. In addition, the sera from infected human patients and micerecognized most of the full length polypeptides of the various proteinsbut did not recognize the cleaved C-terminal proteolytic fragmentspresent in these preparations, which fragments were recognized by ananti-AC monoclonal antibody. Taken together, these results suggest thatthe formation of protective epitopes on AC-Hly and the recognition ofAC-Hly by the sera from infected subjects require the presence of aspecific structure formed only when the modified and repeat regions ofAC-Hly are present. It is conceivable that the formation of thisstructure could require the modification, by acylation, of the fattyacid chains after the translation of AC-Hly at the level of the lysineresidue 983 and could be abolished by the elimination of the C-teminalpart of AC-Hly. In this regard, it is important that both the haemolyticand cytotoxic activities of AC-Hly are lost when the last 75 residues ofAC-Hly, which contain the unmodified secretory signal, are eliminated,and are not directly involved in the activity of formation of pores (4and 29). These observations support, in addition, the hypothesis thatthe extreme part of the C-terminal region of AC-Hly plays an essentialrole in the overall structure of AC-Hly.

It has already been suggested that major protective epitopes of AC-Hlycould be located in the AC portion (Guiso, N., M. Rocancourt, M.Szatanik and J. M. Alonso, 1989, Molec. Pathog. 7:373-380 and Guiso, N.M. Szatanik and M. Rocancourt, 1991, Microb. Pathog. 11:423-431). Theresults presented here show that contamination by AC-Hly fragmentsextending over the modified and repeat regions of AC-Hly might enterinto the production of a protective activity associated with thefragments of the AC domain present in the preparations described in theprior state of the art. Nevertheless, the capacity of these contaminantsin minor quantities to produce a protective activity may be doubtful.Another interpretation might be that, under certain conditions, theregion between amino acids 385 and 450 or 500 could also play a role inthe protective activity of AC-Hly. The structure of this region could bemodified in AC-Hly in the absence of the last 217 amino acid residues orin the absence of acylation. The structure of the N-terminal part of40-50 kDa and the presentation to the immune system could induceprotection when this structure is cleaved in relation to the rest of theAC-Hly molecule in B. pertussis culture supernatants. One explanationfor this hypothesis could be that a monoclonal antibody directed againstthe part of the molecule located between amino acids 385 and 400 is aprotective antibody.

It was also examined whether a correlation exists between the protectiveactivity of a truncated protein obtained from r-AC-Hly in vivo and itsactivity in inducing the synthesis of antibodies which could neutralizethe toxic activity in vitro. Such a correlation was not established.While all the proteins having a protective activity induced thesynthesis of neutralizing antibodies, the protein AHR2, which was not atall protective, induced a strongly neutralizing antibody response. Thissuggests that the presence of neutralizing antibodies in relation to thetoxic activity of AC-Hly is not a reliable measure of the induction ofprotection against infection by B. pertussis.

It is moreover important to note that the protein ΔCla, lacking aportion of the hydrophobic domain, exhibited a protective activity. Thisrecombinant protein is a good candidate for inclusion in acellularvaccines against pertussis since it does not exert any residualcytotoxic activity.

Preparation of a vrg Urea Extract

The vrg urea extracts are prepared from the same 3 phase IV bacterialspecies, that is to say from bacteria no longer expressing any vag genebut expressing the vrg genes. The products of these genes are not yetwell characterized, with the exception of the flagella of B.bronchiseptica. The vrg urea extracts are prepared like the vag ureaextracts. Description of the culture media Dehydrated Bordet Gengoumedium

Composition: 1 liter 5 liters Bordet Gengou medium 30 g 150 g Glycerol10 ml 50 ml Pyrolysed water 1 liter 5 liters Adjust the pH to 7.4

Heat

Autoclave for 15 minutes at 120E

Store at 4EC

At the time of use:

Bordet Gengou is enriched with 15 to 20% sheep or horse blood. Melt thetubes and keep melted at 54EC. Add 2.5 ml of sheep blood to each tube,in a sterile manner. Pour the contents of the tube into a sterile Petridish. Remark:

For the identification of Bordetella pertussis from a nasopharyngealsample, use fresh dishes (maximum 7 days at 4EC)

CSM Agar Medium

To prepare 2 liters of a 10-fold concentrated solution: Sodium hydrogenglutamate 107 g (Ref. Prolabo No. 27872.298) L-Proline 2.4 g (Ref. MerckNo. 7434) NaCl (Ref. Prolabo 25 g No. 27810.295) H₂PO₄ (Ref. 5 g ProlaboNo. 26926.298) KCl (Ref. Prolabo 2 g No. 26759.291) MgCl₂ (Ref. Prolabo1 g No. 25108.295) Tris-base (Ref. Merck 15.2 g No. 8382.2500) Casaminoacids (Ref. Difco 5 g No. 0288-01-2) 1% CaCl₂ solution in 20 mlpyrolysed water (Ref. Prolabo No. 22317.297 Pyrolysed water qs 1 liter

Dissolve the various constituents in a portion of the final volume ofwater. Adjust the pH to 7.4 using hydrochloric acid. Fill to the finalvolume and store at −20EC.

At the time of use, mix:

100 ml of the 10-fold concentrated solution

900 ml of pyrolysed water

1 9 of (2,6-O-dimethyl)cyclodextrin, reference Aldrich No.51166-71-3

15 g of Bacto agar, reference Difco No.0140-01 Distribute, in fractionsof 20 ml, into glass tubes.

Sterilize and add the sterile complement.

Solution of complement:

Mix:

1 ml of solution of complement concentrated 10-fold

100 mg of glutathion, reference Merck No.4090

9 ml of pyrolysed water

Filter this solution on a 0.22 μm Millex filter.

Add 200 μl of this solution to 1 tube of 20 ml of medium.

Stainer Culture Medium

A. Basal Medium

To prepare 2 liters of a 10-fold concentrated solution: Sodium hydrogenglutamate 214.0 g (Ref. Prolabo No. 27872.298) L-Proline (Ref. Merck No.7434) 4.8 g NaCl (Ref. Prolabo No. 27810.295) 50.0 g H₂PO₄ (Ref. 10.0 gProlabo No. 26926.298) KCl (Ref. Prolabo No. 26759.291) 4.0 g MgCl₂(Ref. Prolabo 2.0 g No. 25108.295) Tris-base (Ref. Merck 30.5 g No.8382.2500) 1% CaCl₂ solution in pyrolysed 40 ml water (Ref. Prolabo No.22317.297 Pyrolysed water qs 2 liters

Dissolve the various constituents in a portion of the final volume ofwater. Adjust the pH to 7.6 using hydrochloric acid. Fill to the finalvolume and distribute this concentrated solution which may be stored at−20EC for several weeks.

At the time of use, dilute the medium, sterilize it at 120EC for 15minutes and then add the complement sterilized by filtration.

B. Solution of Complement

To prepare 200 ml of a 10-fold concentrated solution: L-cystine (Ref.Prolabo No.23260.184) 8 g Concentrated HCl 20 ml

Dissolve. Over this preparation, pour the following mixture dissolvedbeforehand: FeSO₄.7H₂O 2 g (Ref. Prolabo No. 24244.232) L (+) ascorbicacid 4 g (Ref. Prolabo No. 20155.237) Nicotinic acid 0.8 g (Ref. MerckNo. 6817) Pyrolysed water 120 ml

Bring to 200 ml with pyrolysed water, distribute the solution infractions of 1, 2, 3 or 4 ml and freeze at −20EC.

At the time of use, dilute the solution 10-fold in pyrolysed water andadd: glutathion (Ref. Merck No.4090) . . . 100 mg/10 ml of dilutedcomplement, sterilize this solution by filtration (0.22 μm Millex filterfor single use) and add 1 ml of sterile solution to 100 ml of sterilebasal medium.

2.1.2. Preparation of the vrg Urea Extract

Centrifuge the bacterial suspension for 30 minutes at 5000 g, at 4EC.

Resuspend the bacterial pellet in 5M urea prepared in PBS buffer(described further on) in an amount of a volume equal to 5 times the wetweight of the bacterial pellet.

Leave stirring for 1 hour at 4EC and then centrifuge for 40 minutes for40,000 g, at 4EC,

Store the supernatant at −80EC until use.

2.1.3. Inactivation of the vrg Urea Extract

After removal of the urea by passage over a G25 column, the vrg ureaextract is diluted in PBS so as to obtain a protein concentration of 300μg/ml.

Add dropwise a volume of glutaraldehyde at 2.5% so as to obtain a finalconcentration of 0.05%.

Leave for 2 hours at room temperature while mixing regularly.

Stop the reaction by addition of lysine (final concentration 0.02M).

After 2 hours at room temperature, the urea extract is adsorbed onaluminium hydroxide prepared in PBS (1 mg/ml), overnight at 4EC, withgentle stirring. The vaccine is then ready for immunization of theanimal in an amount of 10 to 20 μg/injection (described further on).

1-35. (canceled)
 36. A purified nucleic acid comprising a nucleotidesequence encoding an immunogenic polypeptide of adenylcyclase-haemolysin (AC-Hly), which induces formation of protectiveantibodies against an infection by a bacteria selected from the groupconsisting of B. pertussis, B. parapertussis, and B. bronchiseptica whenthe nucleic acid or polypeptide is administered to a human or animalhost, and which is selected from: a) a nucleic acid comprising anucleotide sequence encoding a polypeptide comprising an amino acidsequence from about amino acid 910 or 913 to the last C-terminal aminoacid of AC-Hly from B. pertussis (SEQ ID NO:2) or from B. parapertussisor B. bronchiseptica (SEQ ID NO:4), provided that the polypeptide doesnot include the amino acid residues 1-384 of AC-Hly; b) a nucleic acidcomprising a nucleotide sequence encoding a polypeptide consisting of anamino acid sequence from amino acid 385 to amino acid 400 of AC-Hly fromB. pertussis (SEQ ID NO:2) or from B. parapertussis or B. bronchiseptica(SEQ ID NO:4); c) a nucleic acid comprising a nucleotide sequenceencoding a polypeptide consisting of an amino acid sequence from aminoacid 385 to amino acid 500 of AC-Hly from B. pertussis (SEQ ID NO:2) orfrom B. parapertussis or B. bronchiseptica (SEQ ID NO:4); and d) anucleic acid comprising a nucleotide sequence encoding a polypeptidecomprising the polypeptides defined above in a) and b), the polypeptidesbeing combined via the chain of amino acids naturally present betweenthe polypeptides defined above in a) and b), within AC-Hly from B.pertussis, B. parapertussis, or B. bronchiseptica, or combined via anantigenic sequence from a protein different from AC-Hly, provided thatthe polypeptide does not include the amino acid residues 1-384 ofAC-Hly.
 37. A purified nucleic acid according to claim 36, wherein thepolypeptide encoded by the nucleotide sequence comprises an amino acidsequence from amino acid 385 to the last C-terminal amino acid of AC-Hlyfrom B. pertussis (SEQ ID NO:2) or from B. parapertussis or B.bronchiseptica (SEQ ID NO:4), provided that the polypeptide does notinclude the amino acid residues 1-384 of AC-Hly.
 38. A purified nucleicacid according to claim 36, wherein the polypeptide encoded by thenucleotide sequence comprises an amino acid sequence from amino acid 385to about amino acid 400 of AC-Hly from B. pertussis (SEQ ID NO:2) orfrom B. parapertussis or B. bronchiseptica (SEQ ID NO:4).
 39. A purifiednucleic acid according to claim 36, wherein the polypeptide encoded bythe nucleotide sequence consists of an amino acid sequence from aminoacid 385 to about amino acid 500 of AC-Hly from B. pertussis (SEQ IDNO:2) or from B. parapertussis or B. bronchiseptica (SEQ ID NO:4).
 40. Anucleic acid composition comprising a first and second nucleic acidaccording to claim 36, wherein the first nucleic acid comprises anucleotide sequence from B. pertussis and the second nucleic acidcomprises a nucleotide sequence from B. parapertussis.
 41. A nucleicacid composition comprising a first, second, and third nucleic acidaccording to claim 36, wherein the first nucleic acid comprises anucleotide sequence from B. pertussis, the second nucleic acid comprisesa nucleotide sequence from B. parapertussis, and the third nucleic acidcomprises a nucleotide sequence from B. bronchiseptica.
 42. A nucleicacid composition comprising a first and second nucleic acid according toclaim 36, wherein the first nucleic acid comprises a nucleotide sequencefrom B. parapertussis and the second nucleic acid comprises a nucleotidesequence from B. bronchiseptica.
 43. The purified nucleic acid accordingto claim 36, wherein the nucleic acid comprises a nucleotide sequenceencoding a polypeptide sequence that comprises an amino acid sequencefrom amino acid 910 to the last C-terminal amino acid of AC-Hly,provided that the polypeptide does not include the amino acid residues1-384 of AC-Hly.
 44. The purified nucleic acid according to claim 36,wherein the nucleic acid comprises a nucleotide sequence encoding apolypeptide sequence that comprises an amino acid sequence from aminoacid 913 to the last C-terminal amino acid of AC-Hly, provided that thepolypeptide does not include the amino acid residues 1-384 of AC-Hly.45. The purified nucleic acid according to claim 36, wherein the nucleicacid comprises a nucleotide sequence encoding a polypeptide sequencethat consists of an amino acid sequence from amino acid 385 to aminoacid 400 of AC-Hly.
 46. The purified nucleic acid according to claim 36,wherein the nucleic acid comprises a nucleotide sequence encoding apolypeptide sequence that consists of an amino acid sequence from aminoacid 385 to amino acid 500 of AC-Hly.
 47. The purified nucleic acidaccording to claim 36, wherein the nucleic acid comprises a nucleotidesequence encoding a polypeptide sequence that comprises an amino acidsequence from amino acid 385 to amino acid 500 of AC-Hly.
 48. Thepurified nucleic acid according to claim 36, wherein the nucleic acidcomprises a nucleotide sequence encoding a polypeptide sequence fromamino acid 385 to the last C-terminal amino acid of AC-Hly3, providedthat the polypeptide does not include the amino acid residues 1-384 ofAC-Hly.
 49. A purified nucleic acid comprising a nucleotide sequenceencoding a polypeptide of adenyl cyclase-haemolysin (AC-Hly), whichinduces formation of protective antibodies against an infection by abacteria selected from the group consisting of B. pertussis, B.parapertussis, and B. bronchiseptica when the nucleic acid orpolypeptide is administered to a human or animal host, wherein thenucleic acid comprises a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence from about amino acid 900 or 910 tothe last C-terminal amino acid of AC-Hly from B. pertussis (SEQ ID NO:2)or from B. parapertussis or B. bronchiseptica (SEQ ID NO:4), providedthat the polypeptide does not include the amino acid residues 1-384 ofAC-Hly.
 50. The purified nucleic acid according to claim 48, wherein thenucleic acid comprises a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence from amino acid 900 to the lastC-terminal amino acid of AC-Hly, provided that the polypeptide does notinclude the amino acid residues 1-384 of AC-Hly.